The DNA containing the genes for the oocyte-specific 5S RNA of Xenopus laevis has been purified and extensively characterized. The proposed program takes advantage of our knowledge of the organization of this 5S DNA in an investigation of the relationship of chromatin structure to DNA sequence and regulation of transcription. Studies to date demonstrate that 5S DNA is involved in the nucleosome structure. We shall determine whether particular sequences have a specific location within the nucleosomes in each 700 bp repeat of the DNA, using measurements of susceptibility to staphylococcal nuclease, to restriction endonucleases, and to cross-linking by psoralens. The structure of 5S chromatin in its transcriptionally active and inactive states will be compared in studies on oocytes and somatic cells. An attempt will be made to isolate a chromatin fraction enriched in 5S DNA to allow more detailed studies of its chemical composition. The mechanism of developmental regulation of 5S RNA synthesis will be studied in in vitro transcription experiments involving reconstitution of the template using components from oocytes and somatic chromatins. In a second project, the recombination potential of Xenopus 5S DNA in bacteria will be investigated. Fragments of 5S DNA will be linked to pairs of cloning vectors (plasmids or phage) which have been designed so that recombinants can be selected phenotypically. The recombinants will be characterized by size, by analysis of restriction enzyme digests, and by DNA sequence analysis. In particular, we want to know whether the internal repeats in the AT-rich spacer are sufficiently homologous to cause crossing-over out of exact phase for the whole repeat unit. Such a process has been proposed to explain repeat length heterogeneity in 5S DNA, and as having an important role in the evolution of multigene families. Extensions of this study will be made to other eukaryotic DNAs, and into an investigation of the DNA sequence requirements for recombination in E. coli.